[Structural changes in wild-type Cry3A delta-endotoxin and its mutants in ethyl alcohol solutions at pH 2-2.5].

The methods of circular dichroism, scanning microcalorimetry, electron microscopy, and proteolysis were used to study the ability of wild-type Cry3A-delta-endotoxin and three mutant toxins with cysteine substitutions in helices alpha3 and alpha4 (domain I) to form oligomeric structures in acidic alcohol solutions that reproduce the premembrane environment. At pH 2-2.2 and 20% ethanol, the mutant toxins with single substitutions E132C (alpha3) and E160C (alpha4), as well as the double mutant E132C/S160C with a cysteine bridge connecting helices alpha3 and alpha4, form short linear oligomers specific for Cry3A with a high content of the beta-structure and enhanced sensitivity to proteolysis with pepsin. The data obtained show that the formation of oligomeric structures of this type does not require any divergence of helices alpha3 and alpha4 in domain I of the Cry3A toxin. It has been demonstrated that, at higher pH values in 20% solution of ethanol, the proteins studied are in a metastable state, and their ability to form oligomeric structures depends on temperature.

[Structural changes in Cry3A delta-endotoxin from Bacillus thuringiensis var. Tenebrionis in alcohol solutions at pH 2.0].

Conformational changes in Cry3A delta-endotoxin caused by three different alcohols (ethanol, butanol, and isopropanol) were studied using the methods of circular dichroism, scanning microcalorimetry, and electron miscroscopy. It was shown that, in addition to the standard decrease in the native structure stability, the alcohols can cause a conformational transition that results in a sharp increase in the beta-structure content and a change in the environment of aromatic residues. The conformational transition is accompanied by intermolecular association, which leads to the appearance of oligomers in the form of short filaments. When the alcohols were removed, the oligomers dissociated again into monomers, but it is likely that the native structure either is not restored or is restored only in a small portion of molecules. The oligomer structure is rather cooperative, and its thermostability is higher than that of the initial structure. The disruption of this structure upon heating, observed as a heat absorption peak, is reversible.

[Characteristic of mutants of Pseudomonas putida IPM-36 recombinant strains, selected by anticipating growth on a media containing an inducer of cry3A gene expression]. / Kharakteristika mutantov rekombinantnogo shtamma Pseudomonas putida IPM-36, otobrannykh po priznaku operezhaiushchego rosta na srede s induktorom ékspressii cry3A-gena.

Induction of the expression of the delta-endotoxin gene from Bacillus thuringiensis var. tenebrionis in the recombinant strain Pseudomonas putida IPM-36 negatively affected the viability and the growth rate of the culture. In order to optimize the insecticide production by the recombinant strain, mutant clones exhibiting anticipating growth on an inducer-containing medium were selected and studied. These clones differed in such aspects as the localization of mutations (either in plasmid pBTN11, carrying the cry3A gene, or in the chromosome), growth rate, or the level of delta-endotoxin synthesis after induction. Several mutants obtained proved much superior to P. putida IPM-36 in their structural and segregation stability, although they were as efficient as the original strain with respect to the production of the insecticide (protei Cry3A).

[Features of the production of CryIIIA delta-endotoxin from Bacillus thuringiensis in a recombinant strain of Pseudomonas putida under control of Pm and xylS regulatory elements]. / Osobennosti produktsii CryIIIA delta-éndotoksina Bacillus thuringiensis v rekombinantnom shtamme Pseudomonas putida pod kontrolem reguliatornykh élementov Pm i xylS.

The addition of 3-methyl benzoate to the culture of the recombinant strain Pseudomonas putida IPM-36 (bearing the cryIIIA gene of B. thuringiensis subsp, tenebrionis under the control of the Pm promoter and the regulator gene xylS) slowed down the growth rate of the recombinant strain and increased, under non-selective conditions, the number of plasmid-free cells. Intense synthesis of the Coleoptera-specific delta-endotoxin encoded by the cryIIIA gene began 6-8 h after the addition of the inducer 3-methyl benzoate, no matter whether it was added in the early or late logarithmic phase. Maximal production of endotoxin (0.5-0.6 g/l) was observed when the inducer was added in the early logarithmic phase (3 h of growth). Overproduction of delta-endotoxin impaired cell division, so that filamentous cells became predominant in the culture. delta-Endotoxin accumulated in overproducing cells as irregular crystalloid inclusions.

[Structure and biological features of the temperate phage of Bacillus thuringiensis var. galleriae 69/9, sensitive to chloroform]. / Strukturai biologicheskie osobennosti chuvstvitel'nogo k khloroformu umerennogo faga Bacillus thuringiensis var. galleriae 69/6.

A new temperate bacteriophage designated Px1 has been isolated from the culture of Bacillus thuringiensis var. galleriae 69/6 producing enthobacterin. The bacteriophage belongs to morphological group B1 in accordance with the classification by D. Reanney and H. Ackerman. The bacteriophage head has an isometric multifaceted form with 40 nm diameter. The length of its noncontractile transversely lined tail is 130 nm. High sensitivity to chloroform is peculiar of the phage. The lytical specter of the phage Px1 has been studied. The phage is shown to be capable of efficient transduction of plasmids between the bacteria of Bacillus cereus group.

[Transduction ability of mutants of phage CP51, virulent for bacteria of the Bacillus cereus group]. / Transdutsiruiushchaia sposobnost' mutantov faga CP51, virulentnogo dliia bakterii gruppy Bacillus cereus.

The virulent phage CP51 used usually to transfer chromosomal and plasmid markers between bacteria of the Bacillus cereus group was treated with N-methyl-N'-nitro-N-nitroguanidine. Mutants with reduced viability and ts-mutants were isolated. Some of the mutants were found to have an increased efficiency of transduction and allow to simplify the process. Transfer frequencies of the plasmid pBC16 by the phages CP51-26 and CP51-4-59 were 5 x 10(-4) per plaque-forming unit and 4-5 x 10(-3) per bacterial cell, respectively. Possibilities of further increasing the transduction efficiency of Bacillus thuringiensis genetic material using phage CP51 mutants are discussed.

[General method for selecting mutations in bacterial and phage genes essential for the development of bacteriophage mu]. / Obshchii metod otbora mutatsii v bakterial'nykh i fagovykh genakh, sushchestvennykh dlia razvitiia bakteriogaga Mu.

A method has been developed for a relatively easy selection of bacterial mutations, either inhibiting or promoting Mu growth, and of various mutations in the genome of the Mu phage itself. Spontaneous or induced mutations inhibiting Mu growth are tested in a bacterial strain carrying a Mucts62 prophage in the chromosome and a defective Muc+ prophage within an unstable and frequently eliminated plasmid (the "operational" strain). The cells of such a strain form colonies at 43 degrees C owing to the domination of the c+ allele, though a portion of the cells in a growing colony lose the plasmid with the Muc+ gene and die, dut to the induction of the Mucts prophage. In this case, a large number of phage particles are produced which cause cell lysis during the growth of a colony of the "operational" strain on a Mu-sensitive Escherichia coli lawn. The absence of a lysis zone around a colony of the "operational" strain would indicate a clone that is unable to produce viable Mu particles, as a result of a mutation in the bacterial or the phage genome. The situation is reverse when a strain incapable of maintaining Mu growth is used as the "operational" strain. Clones are tested for the ability to produce Mu on colour indicator media where the "operational" cells form coloured colonies and the tester cells form a colourless lawn. The method has been proved effective in a study conducted to test the use of Mu-lambda-Mu structures with defective lambda prophages flanked by identically oriented Mu genomes for selecting deletion mutants of Mu.

[Detection, on the Escherichia coli chromosome, of the locus necessary to maintaining an autonomous state of sex factor F'lac]. / Obnaruzhenie na khromosome Eschrichia coli lokusa, neobkhodimogo dlia podderzhaniia v avtonomnom sostoianii polovogo faktora F'lac.

The ability of some Escherichia coli strains to serve as effective recipients in crosses with donor strains carrying various plasmids (F'lac, F'lac gal Mu, RP4) has been studied. The experiments have shown that there is a locus maintaining the autonomous state of the F-factors. The function of this locus named Fpm ("F plasmids maintenance") is dispensable for both a bacterial cell itself and RP4 plasmid existence in the cell.

[Effectiveness of plasmid RP4 mobilization of the bacterial chromosome in Escherichia coli strains lysogenic for phages Mu and lambda]. / Issledovanie éffektivnosti mobilizatsii bakterial'noi khromosomy plazmidoi RP4 v lizogennykh po fagam Mu i lambda shtammakh Escherichia coli.

The effect of phage lambda on mobilization of Escherichia coli chromosome mediated by the Mu phage and RP4 plasmid has been studied. The efficiency of bacterial chromosome mobilization is an order of magnitude lower than that in the control strain, monolysogenic for phage Mu provided a termoinducible prophage lambda is located separately from Mu. This efficiency is an order of magnitude higher in comparison with the control strain in case prophage is incorporated in the Mu-lambda-Mu structure and does not inhibit Mu phage development. Thus the effect of bacteriophage Mu on mobilization of E. coli chromosome determined by this phage and RP4 plasmid is shown to be dependent on localization of prophage lambda, as well as on functioning of certain prophage genes. A possibility of replication of Mu genomes from the ori site of prophage lambda incorporated into the Mu-lambda-Mu structure is discussed. This replication is presumed to have a stimulating effect on the Mu-induced mobilization of the bacterial chromosome.

[Genetic study of the effect of lambda phage on Mu phage production in Escherichia coli K-12 strains with Mu--lambda--Mu structures]. / Geneticheskoe issledovanie vliianiia faga liambda na produktsiiu faga Mu v shtammakh Escherichia coli K-12 so strukturami Mu--lambda--Mu.

The interaction of temperate bacteriophages Mu and lambda is studied during their simultaneous induction in specially constructed heterolysogenic strains of Escherichia coli bearing trimeric Mu--lambda--Mu structures. These strains were obtained by the MU-mediated integration of phage lambda circular genomes. Heterolysogenic strains of E. coli were used for studying phage lambda eliminating effect on Mu development with a simultaneous induction of prophages in the same cell. The results of the study allow the localization of the region of phage lambda genome incorporating gene (genes) lambda, which produces an eliminating effect on Mu development.

[Adsorptive capacity of bacteriophage Mu on Escherichia coli K-12 strains with deletions in the attlambda region]. / Izuchenie adsorbtsionnoi sposobnosti bakteriofaga Mu na shtammakh Escherichia coli K-12 s deletsiiami v raione attLambda.

Escherichia coli strains with deletions in att lambda region were obtained. The comparison of the extent of deletions with the sensitivity of the corresponding mutant clones to phage Mu showed that the gene controlling the sensitivity of E. coli K-12 to the phage Mu is located in nad A-gal region of the bacterial chromosome. It is shown that the resistance of E. coli strains which had lost the region of bacterial chromosome between nad A gene and genes of gal-operon have adsorption character. Deletion of the nad A-gal region does not affect the adsorption of other phages (lambda, P1 and T4). Thus, the gene, located in this region, is responsible for the specific adsorption of the phage Mu.